The current research of our group is focused on the mechanism of action of ATP-dependent molecular chaperones and the role of chaperones in ATP-dependent proteolysis. Molecular chaperones are present in all organisms and are highly conserved. They interact with proteins to mediate protein remodeling, folding, unfolding, assembly, and disassembly without themselves being part of the final complex. Chaperones participate in many cellular processes such as DNA replication, regulation of gene expression, cell division, membrane translocation, and protein degradation. Many are induced by environmental stresses and play a critical role during cell stress by preventing the appearance of folding intermediates that lead to irreversibly damaged proteins. They assist in the recovery from stress by refolding and reactivating damaged proteins and by disaggregating aggregated proteins. Some chaperones interact with specific proteolytic components forming compartmentalized ATP-dependent proteases. When associated with proteases, chaperones function in the delivery of damaged proteins and specific regulatory proteins to the proteolytic component. [unreadable] [unreadable] We previously found that Escherichia coli ClpA, an AAA+ ATPase and the regulatory component of ClpAP protease, has molecular chaperone activity. This finding demonstrated that Clp ATPases comprise a family of ATP-dependent chaperones and that molecular chaperones participate directly in proteolysis. One of the aims of our group is to elucidate the mechanism of action of Clp ATPases, including ClpA and ClpX, and their role in proteolysis in combination with ClpP, the proteolytic component of the ClpAP and ClpXP proteases. We are currently investigating how Clp proteins recognize specific substrates. Many substrates contain recognition signals of approximately 10 amino acids located near either the N- or C-terminus of the substrate. By constructing GFP fusion proteins with ClpA and ClpX substrates, we obtained substrates in which the Clp recognition signal was located in the interior of the primary sequence of the proteins. We found that the internal recognition signals were still functional. Our more recent results show that, in the absence of a high affinity peptide recognition signal at the terminus, two elements are important for recognition of GFP-substrate fusion proteins by ClpA. One element is the recognition signal located internally in the fusion protein. The second element is the C-terminal end of the fusion protein. Together these two elements facilitate binding by ClpA and degradation by ClpAP. [unreadable] [unreadable] ClpB, another member of the Clp chaperone family, is able to dissolve protein aggregates and reactivate proteins in conjunction with the DnaK chaperone and its two co-chaperones, DnaJ and GrpE. The mechanism for ClpB function in disaggregation is still undefined, but two models have been suggested. In one model DnaK, DnaJ and GrpE are proposed to loosen polypeptides on the surface of an aggregate and make them available for ClpB binding and unfolding. In a second model, the disaggregation ability of ClpB has been suggested to be via a crowbar mechanism, using the movements of a long coiled-coil domain of ClpB. This crowbar is predicted to loosen the aggregate or break it into smaller aggregates that can then be acted on by the DnaK chaperone system. In order to determine how ClpB works with the DnaK system, we have been using dimeric RepA protein as a model "small aggregate". RepA is an inactive dimer that can be converted to the active monomer through the action of DnaK, DnaJ and GrpE. We have identified conditions where ClpB is required for the monomerization of RepA. In addition, we are using mutants of RepA to decipher ClpB function from the action of the DnaK chaperone system. In order to identify regions in ClpB important for disaggregation we have made amino acid substitutions in ClpB.